Cholinergic nerves provide a predominant neural control of airway smooth muscle cells (ASMCs) through muscarinic receptors. Simulation of these receptors results in the activation of phospholipase C (PLC) and generation of inositol 1,4,5-triphophate (IP3), which induces Ca2+ release through IP3 receptors (IP3Rs). This well-known signaling pathway is important for muscarinic Ca2+ release and contraction in ASMCs. However, our preliminary study, together with previous findings, suggests that muscarinic stimulation may activate ADP-ribosyl cyclase and then produce cyclic ADP-ribose (cADPR), which induces Ca2+ release from the SR by opening RyRs directly and/or indirectly by disassociating FK506 binding protein 12.6 (FKBP12.6) from these Ca2+ release channels, leading to the amplification of muscarinic Ca2+ release and associated contraction in ASMCs. This novel signaling pathway, ADP-ribosyl cyclase-cADPR-FKBP12.6-RyR, may be hyper-functioned in asthma, contributing to the airway muscle Ca2+ and contractile hyperresponsiveness to muscarinic agonists and other numerous spasomogens. To test these hypotheses, we will address the following questions (specific aims): (1) Does cADPR mediate muscarinic Ca2+ release in normal and asthmatic ASMCs? (2) Is FKBP12.6 required for muscarinic Ca2+ release and the target for cADPR in normal and asthmatic ASMCs? and (3) which subtype of RyRs involves muscarinic Ca2+ release and the roles of cADPR and FKBP12.6 in normal and asthmatic ASMCs? These aims will be implemented using simultaneous measurements of Ca2+ sparks or whole-cell [Ca2+]i and membrane currents in freshly disassociated airway myocytes. Genetic manipulations (gene overexpression and knockout), molecular biological and biochemical methods will be also used in this proposal. This study will extend our understanding of the cellular and molecular mechanisms underlying muscarinic Ca2+ release in normal and asthmatic ASMCs, and may also identify novel therapeutic targets for asthma attacks. Since cADPR, FKBP12.6, and/or RyRs are ubiquitously expressed in a variety of cell types, the mechanistic findings from the proposed study will have general biological and pathological significance for various Ca2+-mediated cellular effects.